More on Apparent Fraudulent Nutrition Research from Iran

by Alan R. Gaby, MD

In the July 2019 issue of the Townsend Letter, I expressed concern that a large and growing number of nutrition studies, coming mostly from Iran, have left me wondering whether the research is fraudulent. That is, whether people were writing papers about research that had not actually been conducted. Fraudulent research is an affront to all who value integrity in science, and it has the potential to harm practitioners and patients who rely on its findings.

Since the July editorial was written, the problem seems to have gotten even worse. It has reached the point where, in my mind, the possibility of fraud should be considered in all nutrition research coming from Iran. My previous editorial listed nine characteristics of published papers that might suggest fabricated research. I now add another characteristic: that the research was conducted by a student as part of a graduate school thesis, and that the magnitude of the project seems to have been beyond the capabilities and resources of a student.

This tenth characteristic was added in part because of an article published in the journal Science in 2016. That article, which was titled, “In Iran, a shady market for papers flourishes,” revealed that numerous companies in Iran will write theses and scientific papers for a fee.¹ According to one Iranian scientist, many politicians and diplomats obtain advanced degrees from universities, without actually earning them. Having an advanced degree allows them to compete better for scarce jobs in the science industry, and the law mandates that individuals with these degrees earn a higher salary for the same job. One company offers to have a paper placed in a journal published by Elsevier or Springer for a fee of $1,660. Many young scientists who are unable to find another job are involved in the paper-selling business.

In this editorial, I will review five recently published eyebrow-raising studies from Iran, three of which were conducted by graduate students. It is hoped that these examples will help illustrate the scope of the problem and will also provide the reader with tools to evaluate the credibility of suspect research papers.

Peppermint Oil Gel to Prevent Pressure Sores

A student in Semnan, Iran, as part of a Master of Science thesis, conducted a double-blind trial that examined the effect of topical peppermint oil on the incidence of stage 1 pressure injury (pressure sores) in critically ill patients. One hundred fifty patients admitted to a neurosurgical intensive care unit (ICU) because of head trauma, who required endotracheal intubation, were randomly assigned to have a gel containing peppermint oil or a placebo gel applied to at-risk areas of their skin three times per day.² The incidence of stage 1 pressure injury was significantly lower in the peppermint group than in the placebo group (23% vs. 77%). This study has numerous eyebrow-raising features.

1. Discrepancy related to the number of subjects: The sample size was listed as 150 in the paper and 70 in the document submitted to the Iranian Registry of Clinical Trials (IRCT).

2. Unusually low mortality rate: In most studies of patients with severe head trauma, the 30-day mortality rate was 20-50%. While the peppermint study only followed patients for 14 days, the mortality rate of 1.3% (2 of 150 patients) seems unusually low.

3. Weirdness related to preparing the peppermint gel: The paper stated that the peppermint gel was prepared “according to standard methods,” as described in Appendix A. Briefly, peppermint oil was added to five different concentrations of carbopol 934 in water. Carbopol 934 is a polymer that is used as a thickening and gelling agent. The carbopol 934 concentration that produced the most desirable viscosity (not too runny, not too thick) was used to make the peppermint gel. The 190-word description of this “standard method” was taken verbatim (without attribution) from a 2007 paper on the use of peppermint gel to prevent nipple cracks in breastfeeding women.³ This plagiarism including misspelling of the word “viscous” as “viscose.” Putting aside the issue of plagiarism, one wonders why the researchers bothered to go through the process of testing five different carbopol 934 concentrations, when the 2007 study had already determined which concentration works best.

4. Issues related to the study sites: All four authors listed in the paper were affiliated with Semnan University of Medical Sciences in Semnan, Iran. However, the study itself was conducted at 2 ICUs in Shiraz, Iran, which is more than a nine-hour drive from Semnan. None of the study authors listed an affiliation with any institution in Shiraz. In addition, the study was approved by the ethics committee of Semnan University of Medical Sciences. It is surprising that ICUs in Shiraz, which were treating critically ill patients, would have agreed to conduct a study without first running it by their own ethics committees.

5. Discrepancy related to the outcome measure: The stated outcome measure in the published paper was the incidence of stage 1 pressure injury, as defined by the National Pressure Ulcer Advisory Panel. In contrast, the IRCT registration document stated that the outcome measure was the incidence of pressure ulcer. Stage 1 pressure injury is defined as “intact skin with a localized area of nonblanchable erythema, which may appear differently in darkly pigmented skin.” These lesions are not ulcers, although they may eventually progress to become ulcers.

6. Question related to funding: Because randomized controlled trials are expensive to conduct, such trials are generally reserved for treatments for which there is some evidence of efficacy (such as case reports or uncontrolled trials). Such was not the case with respect to peppermint gel for prevention of pressure injury.

7. Issue related to the assessment method: The paper stated that a pair of observers (nurse plus specialist) examined at-risk areas of the skin (hips, sacrum, back, elbows, knees, heels, and shoulders) once a day for evidence of pressure injuries. It was not stated what was meant by “specialist;” one might assume that referred to the neurologist or neurosurgeon involved in the case. It is difficult to believe that such a “specialist,” who would be evaluating and treating a room full of seriously ill patients, would agree to spend extra time checking the hips, sacrum, back, elbows, knees, heels, and shoulders every day for early evidence of pressure injuries (such would likely be the job of the nurse), particularly when the request to perform such an examination was coming from a graduate student from a faraway university who was studying a treatment for which there was no prior evidence of efficacy.

Pomegranate Juice and Synbiotics for Polycystic Ovary Syndrome

A student in Shiraz, Iran, as part of a master’s of science thesis, conducted a randomized controlled trial that examined the effect of pomegranate juice, a synbiotic beverage (probiotic plus inulin), a beverage containing both pomegranate juice and the synbiotic, or a placebo beverage on various laboratory parameters in women with polycystic ovary syndrome. Ninety-two women consumed their assigned beverage (300 ml per day) for eight weeks. Outcome measures included fasting blood glucose, insulin resistance, and serum levels of lipids, testosterone, luteinizing hormone, follicle stimulating hormone, C-reactive protein, malondialdehyde (a marker of oxidative stress), and serum total antioxidant capacity. Most of these parameters improved in the groups receiving synbiotics (with or without pomegranate juice), whereas there was no clear benefit of pomegranate juice alone. This research generated two papers in the medical literature, each of which presented a different portion of the data.^4,5 That both papers came from the same study is demonstrated by the fact that both listed the same registration document in the Iranian Registry of Clinical Trials (IRCT). These papers raise a number of concerns.

1. Issues related to the beverages: The papers stated that all four of the test drinks were identical in appearance, color, and taste. However, there are several reasons to believe that could not be true. First, the placebo consisted of water, pomegranate flavor, and red food coloring. Iran is the largest producer of pomegranates in the world, so it is likely that many of the study participants knew how pomegranate juice tastes and could distinguish it from artificially flavored, artificially colored water. Second, inulin was added to the synbiotic drinks at a concentration of 20 g/L. At high concentrations in water, inulin forms a gel, and at lower concentrations it has a white, creamy appearance. It seems unlikely that the drinks that contained inulin would look the same as those without inulin. Third, the papers stated that participants consumed 300 ml per day of their assigned beverage, and each person received a two-liter bottle once a week. At a dose of 300 ml per day, a two-liter bottle would leave everyone 100 ml short each week.

2. Discrepancy related to inclusion criteria: One of the inclusion criteria for the study was oligomenorrhea. In the published papers, oligomenorrhea was defined as at least 35 days between menstrual cycles. However, the IRCT registration document defined oligomenorrhea as less than six to nine menses per year.

3. Discrepancy related to recruitment dates: The published research stated that recruitment began in January 2017. However, the IRCT document stated that recruitment started on February 19, 2017.

4. Discrepancies related to outcome data: The IRCT document listed serum cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides as outcome measures. It did not mention fasting blood glucose, insulin sensitivity, malondialdehyde, total antioxidant capacity, C-reactive protein, testosterone, luteinizing hormone, or follicle stimulating hormone, even though the IRCT document was updated after the study was completed.

5. Questions related to logistics: Both papers stated that the study drinks were prepared fresh once a week and were delivered to participants immediately after they were prepared. However, one of the papers also stated that, in order to assess treatment compliance, participants were asked to return their empty bottles “when picking up their beverages for the following week.” So, which was it? Did the researchers deliver the bottles, or did the patients have to come and pick them up each week? It seems like a daunting task for a graduate student to make 92 deliveries relatively “immediately” in a city of 1.6 million people where there are significant problems with traffic congestion. It is also difficult to believe that people would agree to participate in a study that required them to navigate city traffic each week to pick up a bottle of juice or placebo.

6. Discrepancy related to the probiotics used: One of the papers⁴ stated that each liter of synbiotic beverage contained 2 x 10⁸ colony-forming units (CFU)/g of lactobacillus. However, several paragraphs later, it stated that the synbiotic beverage contained 1 x 10⁸ CFU/ml of each of 3 different strains: Lactobacillus rhamnosus GG, Bacillus koagolans, and Bacillus indicous. Bacilli are not the same as lactobacilli, so these statements are incompatible.

L-Carnitine for Pemphigus Vulgaris

A student in Tehran, Iran, as part of a master’s of science thesis, conducted a double-blind trial that examined the effect of L-carnitine (2 g per day for 8 weeks) on various laboratory parameters in patients with pemphigus vulgaris. Outcome measures included serum levels of LDL cholesterol and triglycerides, total oxidant capacity, total antioxidant capacity, serum markers of bone turnover (osteopontin and bone morphogenic protein) and a serum marker of renal function (cystatin C). Most of these parameters improved significantly in the L-carnitine group compared with the placebo group. This research generated two papers in the medical literature, one in 2018⁶ and one in 2019.⁷ That both papers came from the same study is demonstrated by the fact that both listed the same registration document in the Iranian Registry of Clinical Trials (IRCT). Each paper presented a different portion of the data. Examination of these papers raises several issues.

1. Discrepancy in sample size: One paper stated that 52 patients were randomized, whereas the other paper stated that 48 were randomized. This difference was due to a difference in the number of eligible patients who refused to participate.

2. Discrepancies regarding inclusion criteria: The 2018 paper stated that patients had to have pemphigus vulgaris for at least one year. The 2019 paper stated that patients were eligible if they had the disease for “about 1 year.” Considering that pemphigus vulgaris is a chronic disease, these different inclusion criteria would result in very different patient populations.

3. Discrepancy in compliance assessment: One paper stated that compliance was determined by measuring serum carnitine levels, whereas the other paper stated that compliance was assessed by the number of pills returned.

4. Discrepancy in the number of withdrawals: One paper stated that four patients withdrew in the L-carnitine group and six withdrew in the placebo group. The other paper stated that six patients withdrew in the L-carnitine group and no patient withdrew in the placebo group.

5. Discrepancy in the source of the study medication: Both papers stated that L-carnitine and placebo were manufactured by Asal Daroo Kish Pharmaceutical Company in Kish, Iran. The IRCT document stated that L-carnitine and placebo were manufactured by the KAREN Chemistry Company.

6. Issues related to outcome measures: Two of the outcome measures used in the 2019 paper (bone morphogenic protein and cystatin C) were added to the IRCT document more than two years after the trial was completed. It is possible that the researchers used stored frozen serum to measure these parameters. However, when researchers use stored samples from a previous study, they typically indicate so in the Materials and Methods section, which was not done in this case.

7. Question related to funding: Because randomized controlled trials that include multiple outcome measures are expensive to conduct, such trials are generally reserved for treatments for which there is some evidence of efficacy (such as case reports or uncontrolled trials). Such was not the case with respect to L-carnitine as a treatment for pemphigus vulgaris. One wonders why Tehran University of Medical Sciences, which almost certainly has limited financial resources, would have funded such a study.

8. The day before this editorial was completed, a third paper based on the same L-carnitine/pemphigus study appeared in the medical literature. In this paper, new lab tests were conducted, including serum levels of secreted frizzled‐related protein‐5 (SFRP5), omentin, and visfatin.⁸ These outcome measures were added to the IRCT document more than two years after the trial was completed. Similar to point number 6 (above), no mention was made that stored frozen samples were being used for these additional lab tests. Even more baffling was the statement that the sample size for the study was calculated based on the standard deviation and difference in mean for serum SFRP5 (those values had been reported previously in the medical literature). There are two problems with that statement. First, one wonders why the Iranian researchers would have based their sample size on serum SFRP5 data, considering that they did not decide to measure that variable until two years after the study was completed. Second, the statement contradicts one of the other papers, which stated that the sample size was based on the standard deviation and mean difference of serum osteopontin (not SFRP5).

Vitamin E Vaginal Suppositories for Atrophic Vaginitis

In the November 2019 issue of the Townsend Letter I reviewed a 2019 paper that found vitamin E vaginal suppositories were as effective as conjugated estrogens vaginal cream for improving sexual function in postmenopausal Iranian women with vaginal atrophy.⁹ I commented at the time that it is difficult to believe vitamin E would be as effective as conjugated estrogens, and that confirmatory studies are needed. I have since learned that another paper, from 2016, came from this same research (both papers listed the same IRCT document and the papers had many identical features). The 2016 paper reported that vitamin E vaginal suppositories improved histological signs of vaginal atrophy in postmenopausal women, although it was somewhat less effective than intravaginal estrogen.¹⁰ Examination of these papers together raised several additional concerns.

1. Discrepancy in recruitment dates: One paper said the study was conducted in 2014, whereas the other paper said it was conducted from March 2013 to April 2014.

2. The score on the Abbreviated Sexual Function Questionnaire was listed as the primary outcome measure in the 2019 paper. However, this questionnaire was not mentioned as one of the outcome measures in the IRCT document.

3. Discrepancies in the inclusion criteria: One paper listed an elevated serum FSH level as an inclusion criterion; the other paper did not. One paper listed a six-week interval since bilateral oophorectomy; the other paper did not. One paper listed symptoms of vaginal atrophy based on patient self-report; the other paper listed vaginal atrophy based on patient complaints and researcher’s examination. One paper listed additional specific criteria for women aged 40-50 years; the other paper did not.

4. Study participants were required to submit to four Pap smears over a period of 12 weeks. It is difficult to believe that many women would agree to participate in such a study.

Vitamin C and Coronary Artery Bypass Grafting Surgery

Fifty patients in Shiraz, Iran, who were undergoing coronary artery bypass grafting surgery were randomly assigned to receive, in double-blind fashion, vitamin C or placebo. Patients in the vitamin C group received 5 g of vitamin C intravenously before induction of anesthesia and an additional 5 g of vitamin C in the cardioplegic solution. The placebo group received normal saline. Compared with placebo, vitamin C significantly improved left ventricular ejection fraction at 72 hours after surgery and significantly decreased the length of stay in the intensive care unit.¹¹ After reviewing this paper, I found it difficult to believe that such a study would have actually been conducted.

The main concern surrounding this study is that adding a large dose of vitamin C to a cardioplegic solution has not been demonstrated to be safe, and it has the theoretical potential to cause serious adverse effects. Cardioplegic solutions are used to stop the heart during open heart surgery. They typically consist of electrolytes such as sodium, potassium, magnesium, and calcium, and have an osmolarity similar to that of serum. Commercially available cardioplegic solutions are mildly acidic and must be adjusted to a pH of 7.4 to 7.8 before use, by adding 10 ml of 8.4% sodium bicarbonate to each liter of cardioplegic solution. The addition of vitamin C to a cardioplegic solution would increase its osmolarity, making it a hypertonic solution. It would also decrease the pH of the sodium bicarbonate-adjusted solution. The extent of that pH reduction would depend on the pH of the injectable vitamin C, which typically varies from 5.5 to 7.0 in commercial products. It would also depend on the volume of the cardioplegic solution to which the vitamin C was added, but that information was not provided in the paper. Nor was there a mention of any attempt to adjust the pH of the cardioplegic solution back to 7.4 to 7.8 after vitamin C was added. These changes in osmolarity and pH of the cardioplegic solution could conceivably interfere with the attempt to restart the heart.

A literature search revealed only one study that addressed the safety of adding vitamin C to a cardioplegic solution. That was a 1989 study that found no adverse effects of ascorbate in an animal model of cardiopulmonary bypass and ischemic cardiac arrest. It is not clear what the vitamin C concentration was in the cardioplegic solution used in the Iranian study, because the researchers did not state what volume of cardioplegic solution was mixed with 5 g of vitamin C. Let’s assume the volume was 1 liter, because many commercial cardioplegic solutions come in 1-liter bags. If that is the case, then the concentration of vitamin C used in the Iranian study was around 2.8 times higher than the concentration that was investigated in the 1989 animal study.

One can only imagine what was in the “informed consent” form that the patients signed. Perhaps it was something like, “We are going to stop your heart during your open-heart surgery by using a standard heart-stopping solution. There is a 50% chance we will add a large amount of vitamin C to the heart-stopping solution. We have no idea whether adding vitamin C will affect our ability to restart your heart, because that has never been studied in humans. But our study is in the interest of science, so please sign here.”

Final Remarks

Honest scientists in Iran, in order to protect their own reputations, should help investigate questionable research and speak out against it. Journal editors and publishers also have an important role to play. First, they should insist on more rigorous peer review of submitted papers. It is astounding that peer reviewers failed to identify any of the red flags that I described in this editorial. Second, journal editors must overcome their resistance to investigating allegations of possible scientific misconduct. All too often, editors have seemed willing to accept a flimsy or factually incorrect response from the authors of questionable papers, rather than putting in the time and effort needed to investigate the allegations of “whistleblowers.” The future of biomedical sciences depends on a major cleanup effort.